Colorectal cancer (CRC) is currently the third leading cause for cancer-related mortality. Cancer stem cells have been implicated in colorectal tumor growth, but their specific role in tumor biology, including metastasis, is still uncertain. Increased expression of L1CAM, CXCR4 and NODAL was identified in tumor section of patients with CRC and in patients-derived-organoids (PDOs). The expression of L1CAM, CXCR4 and NODAL was evaluated using quantitative real-time PCR, western blotting, immunofluorescence, immunohistochemistry and flow cytometry. The effects of the L1CAM, CXCR4 and NODAL on tumor growth, proliferation, migration, invasion, colony-formation ability, metastasis and chemoresistance were investigated both and . We found that human colorectal cancer tissue contains cancer stem cells defined by L1CAM/CXCR4 expression that is activated by Nodal in hypoxic microenvironment. This L1CAM/CXCR4 population is tumorigenic, highly resistant to standard chemotherapy, and determines the metastatic phenotype of the individual tumor. Depletion of the L1CAM/CXCR4 population drastically reduces the tumorigenic potential and the metastatic phenotype of colorectal tumors. In conclusion, we demonstrated that a subpopulation of migrating L1CAM/CXCR4 is essential for tumor progression. Together, these findings suggest that strategies aimed at modulating the Nodal signaling could have important clinical applications to inhibit colorectal cancer-derived metastasis.